fragment-based drug discovery applied to hsp90. discovery of

pubs.acs.org/jmc Published on Web 07/27/2010 r 2010 American Chemical Society

5942 J. Med. Chem. 2010, 53, 5942–5955

DOI: 10.1021/jm100059d

Fragment-Based Drug Discovery Applied to Hsp90. Discovery of Two Lead Series with

High Ligand Efficiency†

Christopher W. Murray,* Maria G. Carr, Owen Callaghan, Gianni Chessari, Miles Congreve, Suzanna Cowan,Joseph E. Coyle, Robert Downham, Eva Figueroa, Martyn Frederickson, Brent Graham, Rachel McMenamin,M. Alistair O’Brien, Sahil Patel, Theresa R. Phillips, Glyn Williams, Andrew J. Woodhead, and Alison J.-A. Woolford

Astex Therapeutics, Ltd., 436 Cambridge Science Park, Milton Road, Cambridge CB4 0QA, U.K.

Received January 15, 2010

Inhibitors of the chaperone Hsp90 are potentially useful as chemotherapeutic agents in cancer. Thispaper describes an application of fragment screening to Hsp90 using a combination of NMR and highthroughput X-ray crystallography. The screening identified an aminopyrimidine with affinity in thehigh micromolar range and subsequent structure-based design allowed its optimization into a lownanomolar series with good ligand efficiency. A phenolic chemotype was also identified in fragmentscreening and was found to bind with affinity close to 1 mM. This fragment was optimized usingstructure based design into a resorcinol lead which has subnanomolar affinity for Hsp90, excellent cellpotency, and good ligand efficiency. This fragment to lead campaign improved affinity for Hsp90 byover 1000000-fold with the addition of only six heavy atoms. The companion paper (DOI: 10.1021/jm100060b) describes how the resorcinol lead was optimized into a compound that is now in clinicaltrials for the treatment of cancer.

Introduction

Molecular chaperones are proteins that play a role in theconformational stability,maturation, and function of other sub-strate proteins, which are known as clients.1 Heat shock protein90 (Hsp90a) is amolecular chaperone, andmanyof its clients areoncology targets that are known to play critical roles in cancerprogression.2,3 In fact it has been argued that interference withHsp90 and its associated clients can allow the simultaneoustargeting of the hallmarks of cancer, i.e., self-sufficiency ingrowth signals, insensitivity to antigrowth and apoptotic signal-ing, angiogenesis, limitless replicative potential, invasion, andmetastasis.2,4,5 It was initially thought that inhibition of theHsp90 chaperone cycle would lead to unacceptable toxicitiesdue to the ubiquitous expression of Hsp90 in normal cells.6

However, later work has shown that Hsp90 inhibition can leadto reasonably specific inhibition in cancer cells because Hsp90 ispresent in an activated form.6,7 Additionally, the preclinical and,more recently, clinical activity of geldanamycin-based inhibitorshas indicated the potential utility of Hsp90 inhibitors.2,8

The chaperone cycle ofHsp90 involves the turnover ofATPto ADP through an ATPase activity associated with theN-terminal domain ofHsp90.9 TheATPbinding site has beencharacterized crystallographically, and the bindingmodes of a

number of Hsp90 inhibitors have been determined.9 It has alsobeen demonstrated that inhibition leads directly to the down-regulation of client proteins and antiproliferative activity.10 Atthe start of our Hsp90 program the most advanced Hsp90 inhi-bitors were derived from natural products11-15 (see Figure 1)and our aimwas to use fragment-based drug discovery (FBDD)to derive a synthetic inhibitor of Hsp90 with improved pharma-ceutical properties. There are now a number of reviews describ-ing syntheticHsp90 inhibitors,16-19 andFigure 2gives structuresof compounds currently in clinical development.20-25

Fragment-based drug discovery (FBDD) is gathering mo-mentum in the pharmaceutical industry, and a number ofclinical candidates or advanced leads have been identified viathe approach.26-34 A recent review has listed the key conceptsthat lie behind the rise in FBDD, and we recapitulate these toprovide some background to the field. The first concept is thatinappropriate physical properties are thought to be a majordriver for attrition in drug development,35-38 and there is

Figure 1. Natural product derived inhibitors of Hsp90. Note that17-DMAG, 17AAG, and the quinol form of 17AAG (i.e., IPI-504)have been progressed into clinical trials.

†Coordinates for the Hsp90 complexes with compounds 1, 3, 4, 2þ 4, 6,14, 21, 24, and 31 have been deposited in the Protein Data Bank (PDB)under accession codes 2xdk, 2xdl, 2xdu, 2xds, 2xdx, 2xhr, 2xht, 2xhx, and2xab, respectively, together with the corresponding structure factor files.

*To whom correspondence should be addressed. Phone: þ44 (0)1223226228.Faxþ44 (0)1223226201.E-mail: [email protected].

aAbbreviations: CDK4, cyclin dependent kinase 4; FBDD, fragment-based drug discovery; Hsp70, heat shock protein 70; Hsp90, heat shockprotein 90; ITC, isothermal titration calorimetry; LE, ligand efficiency;PDB, Protein Data Bank.

Article Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 5943

therefore a need to explore alternative methods, such asFBDD, that might deliver candidates with improved physicalproperties. It will takemany FBDDapplications acrossmanydifferent targets before progress in this regard can be properlyassessed. Fragments are compounds of low molecular weight(usually 100-250 Da) and typically have low binding affi-nities (>100 μM), but a second key concept is that fragmentsform high-quality interactions despite their weak potency andtherefore can provide attractive starting points for medicinalchemistry. In our application we will show that this is true forHsp90, although no single application can be used to assess ageneral point. A third concept important to FBDD is thatligand efficiency (LE)39,40 can be used to track the potency offragment hits during lead identification and to assess whethergains in potency are significant enough to justify increases inmolecular size. LE can be defined as

LE ¼ -ΔG

HAC¼ -RT lnðKdÞ

HAC

where ΔG is the free energy of binding of the ligand for aspecific protein, HAC is the number of heavy atoms in theligand, and Kd represents the dissociation constant for theprotein-ligand complex (IC50 is often used instead of Kd).This paper illustrates the use of LE during fragment optimiza-tion against Hsp90 but also illustrates that LE is not the onlycriteria that should be used to select which fragments tooptimize. The fourth concept behind FBDD is that relativelysmall libraries of fragments are required to sample chemicalspace and some elegant models have been described in theliterature to illustrate this behavior.41,42 Here no analysis ofthe sampling advantages of fragments is presented but ourapplication does show the identification of two distinct che-mical leads for Hsp90 derived from screening a library of only1600 fragments.

The motivation of this paper is to describe how biophysicalmethods (NMRandX-ray crystallography) have been used toperform fragment screening against theN-terminal domain ofHsp90. The paper shows how two fragment hits were then

efficiently optimized into potent lead series. The results arediscussed together with any learning points that are relevantto either Hsp90 inhibition or FBDD. A companion paper(DOI: 10.1021/jm100060b) describes how one of these leadswas optimized into an Hsp90 inhibitor that is currently inclinical trials for the treatment of cancer.

Results and Discussion

Fragment Screening for Hsp90.Approximately 1600 com-pounds from our fragment library43 were screened in cock-tails using ligand observed NMR via water LOGSY.44

Compounds showing a medium or strong LOGSY signal(as defined in the Experimental Section) were further char-acterized according to competition for the nucleotide site.NMR screening was carried out in the presence of a lowconcentration of the product, ADP, which binds weakly tothe ATP-ase domain of Hsp90 under the screening condi-tions. Cocktails that contained fragments that bound to thenucleotide site of the target could be identified immediatelyby observing the displacement of ADP, estimated from thereduction in its own LOGSY signal. Further information onthe affinity of the fragment at this site was obtained in asecond step by adding 5 mMMg2þ to the screening solution.This increases the affinity ofADP for the active site and leadsto the displacement of more weakly bound fragments. Atotal of 125 fragments were progressed into crystallographyafter analysis of the NMR data and a consideration ofchemical diversity. Of these compounds 26 fragment crystalstructures were obtained, spanning a variety of chemotypes.The chemical structures for four of the validated hits areshown in Figure 3.

All affinities given in this paper are dissociation constantsdetermined by isothermal titration calorimetry (ITC) (seeExperimental Section for more details and for ITC valuesobtained with reference compounds). All ligand efficienciesare calculated directly from the ITC dissociation constantsand have units of kcal per heavy atom.

Figure 2. Synthetic inhibitors of Hsp90 currently in clinical trails.

5944 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 Murray et al.

Compound 1has ameasured affinity of 250μMand ligandefficiency of 0.38. The binding mode for this fragment isgiven inFigure 4a. Themolecule lies in a deep pocketwhere itforms an extensive network of hydrogen bonds with the sidechain of Asp93 and four crystallographically observed watermolecules trapped at the bottom of the pocket. The amino-pyrimidine interactions are similar to those observed withthe adenine ring in the ADP complex (Figure 4b). Interest-ingly, compound 1 is twisted about the bond connecting thepyridine to the pyrimidine. As discussed in more detailbelow, this is not the most stable geometry in the absence

of the protein, so it should be possible to improve the affinitythrough stabilization of the protein bound conformation.Furthermore, compound 1 is doing a poor job of fillingthe proximal lipophilic pocket lined by the lipophilic sidechains of Met98, Leu107, Phe138, Val150, and Val186 (seeFigure 4c), and so improved hydrophobic fit in this regionmay be advantageous.

Compound 2 is an example of an even simpler aminopyr-imidine that was observed to bind crystallographically eventhough it has very weak affinity (>1 mM). Figure 4d dis-plays the Fo - Fc unrefined electron density map for thiscomplex, and despite its small size and weak affinity, theaminopyridine can be unambiguously placed within the maptogether with a number of density peaks associated withtightly bound water molecules. Comparison of Figure 4dwith Figure 4b shows that the simpler aminopyrimidineforms the same contacts as compound 1.

Compound 3 has an affinity of 790 μM and a ligandefficiency (LE) of 0.26 and is a known drug (ethamivan)with respiratory stimulant activity.45 Figure 5a shows thebinding mode in a similar frame of reference to compound 1

in Figure 4b. It can be seen that the phenolic OH ofcompound 3 displaces one of the water molecules observed

Figure 3. Chemical structures of four validated hits identified byfragment screening against Hsp90.

Figure 4. Binding mode of aminopyrimidine fragments in Hsp90. (a) Compound 1 forms multiple hydrogen bonds with water molecules andthe side chain of Asp93 at the bottom of the ATP binding site. The bond between the two aromatic rings is heavily twisted. (b) Thecrystallographic overlays of compound 1 and ADP illustrate the conserved nature of the interactions. (c) Protein surface in gray and the ligandsurface in cyan for the complex of Hsp90 and compound 1. For clarity the only protein residues shown are the residues that line the proximallipophilic pocket (Met98, Leu107, Phe138, Val150, and Val186). The bulge on the middle-right-hand side of the protein surface represents theproximal lipophilic pocket. It is poorly filled by the ligand surface and is not occupied by water molecules. (d) Fo - Fc electron density mapcontoured at 4σ for compound 2. The view has been clipped to aid visualization.


in the pyrimidine/purine structures and forms a hydrogenbond with one of the remaining conserved water molecules.The carbonyl of compound 3 forms a hydrogen bond withthe side chain of Thr184 and with a conserved water mole-cule (mediating to Asp93), but there is no direct hydrogenbond toAsp93.Despite its poor ligand efficiency, the crystal-lographic binding mode offered the promise of a fast frag-ment optimization campaign. Superimposition of the stru-cture on the yeast Hsp90 complex with the natural product,radicicol46 (Figure 5b) indicated that conversion of thephenol to the corresponding resorcinol would allow theformation of a direct hydrogen bond to Asp93 and anadditional water-mediated hydrogen bond. These interac-tions are common to all known active site inhibitors ofHsp90, andwe reasoned that this would yield a large increasein affinity and ligand efficiency. The protein-ligand com-plex also suggested another potential route to fragmentoptimization. The methoxy group of fragment 3 does notproperly fill the proximal lipophilic pocket defined by theside chains of Met98, Leu107, Phe138, Val150, and Val186(Figure 5c), and its replacement with other substituentsmight also be beneficial.

Compound 4 has an affinity >1 mM with an LE<0.34,and it binds in a lipophilic pocket away from Asp93. Theformation of the pocket is driven by the substantial

rearrangement of residues Ile110-Gly114 which becomethe central portion of a long helix. Figure 6a illustrates thischange in protein secondary structure by superimposing thestructures for fragments 2 and 4. The conformationalmovement has previously been observed with larger com-pounds in Hsp9047 and has also been reported with frag-ments, although noX-ray structures of fragments have beendisclosed.48,49 Other conformations for this mobile regionwere also observed during ourHsp90 project, but this one isnotable because it opens up a large lipophilic pocket thatcan be exploited in drug design. Fragment 4 makes onlylipophilic interactions with the induced pocket formed bythe side chains of Leu107, Phe 138, Tyr139, and Trp162.Figure 6b shows the result of an experiment where com-pounds 2 and 4 were cosoaked into Hsp90 that resulted inboth compounds being observed to bind at the same time.The fragment binding modes in the individual fragmentcomplexes are identical to the binding modes observedin the cosoak. Superimposition of the cosoak structure of2 þ 4 with other structures indicates that fragment 4

occupies a region that is filled by the side chain of Leu107in the fragment 2 complex. A valid design approach wouldbe to attempt to link together fragments 2 and 4, but the restof this paper focuses on a fragment growth strategy startingfrom either compound 1 or compound 3.

Figure 5. Binding mode of phenol fragment 3 in Hsp90. (a) Compound 3 displaces one of the water molecules (compare with Figure 4a andFigure 4b) and forms hydrogen bonds with two of the conserved water molecules at the bottom of the ATP binding site. (b) Crystallographicoverlay of compound 3 with the natural product radicicol (PDB code 1BGQ). The additional hydroxyl group in radicicol makes a directhydrogen bond with Asp93 and with a conserved water molecule. (c) Protein surface in gray and the ligand surface in cyan for the complex ofHsp90 and compound 3. For clarity, the only protein residues shown are the residues that line the proximal lipophilic pocket (Met98, Leu107,Phe138, Val150, and Val186). The bulge on the lower-right-hand side of the protein surface represents the proximal lipophilic pocket. It ispoorly filled by the ligand surface and is not occupied by water molecules.


Optimization of Aminopyrimidines Starting fromFragment 1.

Tables 1 and 2 give data and chemical structures for the amino-pyrimidines covered in this section. The tables give dissociationconstants obtained with isothermal titration calorimetry, andthe cell IC50 values reflect the inhibition of HCT116 cellproliferation.

The crystal structure of Hsp90 with compound 1 showsthat the fragment is twisted about the bond connecting thepyridine to the pyrimidine (torsion angle of 47.6�) despite thepresence of an aromatic nitrogen atom ortho to this bond.Small molecule crystal structures50 and the torsion profilereported in Figure 7a both suggest that the optimal geometryfor such ring systems is close to planarity, so it should bepossible to improve the affinity through stabilization of theprotein bound conformation.

Optimisation of fragment 1 beganwith virtual screening ofclose analogues and resulted in the purchase of the simplechloro analogue, compound 5, that showed an improvementin affinity of approximately 100-fold. Initial synthesisfocused on analogues of compound 5 with the aim of stabi-lizing the twist observed in the X-ray structure and filling theproximal lipophilic pocket that is formed by lipophilic sidechains of Met98, Leu107, Phe138, Val 150, and Val186.From the crystal structure of fragment 1, the elegant way

to achieve this was to substitute the upper phenyl ring at the2 and/or 6 position with small groups (Figure 4c).

The first compound synthesized was the 2-methoxy ana-logue 6, which showed a 5-fold improvement in affinity and acrystal structure showed that the compound bound in thedesired conformation (coordinates have been deposited withthe PDB). However, there was a concern that this was stillnot the most favored ligand conformation. The torsionenergy profile of 6 indicated that the methoxy group mightprefer to sit on the other side of ring to avoid a clash with thepyrimidine nitrogen and that the conformation observed inthe crystal structure (torsion angle of 48.1�) is a local mini-mum which is ∼3 kcal/mol higher in energy than the globalminimum (see Figure 7b). This reasoning was supported byfurther SAR. For example, the 2,6-dimethoxy compound 7

offered a further 5-fold improvement over compound 6

despite the fact that the second methoxy group made nodirect contact with the enzyme. Additionally the 2-chlorocompound 9 was significantly more potent than compound5. The torsion profile of 9 suggests that mutating themethoxy groupwith a chloro reduces to only∼1 kcal/mol the

Figure 6. (a) Crystallographic complex of Hsp90 with 4 (in cyan) superimposed on the complex with 2 (in orange). The proteins are displayedin schematic form. The upper left helix is continuous with fragment 4 but is interrupted in a more typical complex with fragment 2. Thissubstantial conformational movement is induced by the binding of fragment 4. (b) Binding mode of fragments 2 and 4 when cosoaked withHsp90. Fragment 4 binds in an induced, slot-shaped, hydrophobic pocket flanked by the lipophilic side chains of Leu107, Phe138, Tyr139,and Tyr162.

Table 1. Substitution with Small Groups at R1 and R2 EnhancesPotency of the Aminopyrimidines

compd R2 R6 ITC (μM) LE cell IC50 (μM)

1a 250 0.38

5 H H 2 0.56

6 OMe H 0.35 0.55

7 OMe OMe 0.068 0.54 7.9

8 Cl OMe 0.036 0.6 6.4

9 Cl H 0.083 0.64 18aThe chemical structure for 1 is shown in Figure 3

Table 2. Further SAR around the Upper Phenyl Ring of the Amino-pyrimidines


difference in energy between local and the global minimum(see Figure 7c). Finally, the 2-chloro-6-methoxy compound 8onlymarginally improves upon this affinity and the torsionaldistribution for 8 shows that all conformationswith a torsionangle between 60� and 110� are isoenergetic (see Figure 7d).Generally the calculated torsion distributions provided use-ful predictions of the activity changes observed in the initialround of synthesis.

Compound 9 was the most ligand efficient molecule fromthe initial synthesis iteration and was chosen as the basis forthe next iteration. Crystal structures on this series indicatedthat further substitution of the upper phenyl ring with smallgroups at the 4-position might facilitate additional lipophilicinteractions with the enzyme. Methoxy or chlorine substitu-tion significantly increased affinity with compound 10 hav-ing an affinity of 12 nM and very high ligand efficiency(0.68).

Further work on this series was focused on trying toimprove the physical properties and the cell activity ofcompound 10. The structural work suggested that substitu-tion at the 5 position of the upper phenyl ring might be usedto allow the introduction of solubilizing groups with the aimof improving cell activity. The 5-methoxy group 12 offered a2-fold improvement in measured potency (although notethat the error in ITC determination is approximately 2-fold).It also served as a growth point for the introduction ofsolubilizing groups such as the morpholine group exempli-fied in compound 14. This compound has low micromolar

activity in the cell assay andmay be a potential leadmoleculeversusHsp90. The crystal structure of compound 14 is shownin Figure 8 and is in good agreement with the designhypotheses; the superimposition in Figure 8 also illustratesthat the final molecule forms the same interactions as theinitial starting fragment.

Optimization of Phenols Starting from Fragment 3. Thebinding mode of fragment 3 indicated that the methoxy

Figure 7. Energy profiles and population histograms of compounds 1, 6, 9, and 12. The continuous line on the plots gives the relative potentialenergy calculated byB3LYP/6-311G* as a function of the torsion angle for each structure. The scale on the right-hand side of the plots gives therelative potential energies in kcal/mol. The relative population of each torsion angle in the gas phase is estimated from the Boltzmanndistribution and is shown as a histogram with the percentage population shown on the left-hand scale in the plot. The torsional angle beingconsidered is highlighted in bold in the chemical structures.

Figure 8. Crystallographic complex of Hsp90 with 14 (in cyan)superimposed on the complex with 1 (in orange). The originalbinding mode of the fragment is maintained in the final complex.


group could be replaced by substituents which filled theproximal lipophilic pocket. Chloro (15), ethyl (16), isopropyl(17), and tert-butyl (18) analogues were initially synthesized(Table 3). The chloro group showed no improvement, whilethe ethyl group showed a 20-fold improvement. The bestgroups were tert-butyl and isopropyl, which were approxi-mately 100-fold more potent. The superior filling of theproximal lipophilic pocket was confirmed by crystallogra-phy on compound 18 (data not shown).

Radicicol (Figure 1) is a resorcinol and contains an addi-tional OH group at the 2-position compared with 4-OH

substituted compounds. Given that this 2-OH group inradicicol forms a direct hydrogen bond to Asp93, we wereinterested to see whether a phenol based on this substitutionpattern would have superior affinity. Compounds 19 and 20

were prepared, but these were about 10-fold less potent thanthe corresponding 4-OH substituted compounds. This im-plied that the 4-OH group must be a significant contributorto the affinity of the series, presumably derived from thefavorable displacement of a mediating water molecule ob-served in the ADP and pyrimidine complexes (compareFigure 5a with Figure 4b).

The next synthetic iteration investigated a selection ofamides to replace the diethylamide in compound 18. TheX-ray structure indicated the importance of the carbonylgroup which forms a direct hydrogen bond with the sidechain of Thr184 and a strongwater-mediated hydrogen bondwithAsp93 (seeFigure 5a). The torsion between the carbonyland phenyl ring is highly twisted (61� in compound 18), andthe torsion is stabilized by the tertiary nature of the amide.The design therefore focused on tertiary amides, and model-ing indicated that they would be directed toward the sidechain of Lys58 which is well-defined in the electron density(Lys58 is visible in the upper-left-hand side of Figure 5a andFigure 5b). The first design idea was to form a hydrogenbond with the amine on Lys58. Compounds 21, 22, and 23

are representative of a small number of compounds that testthis idea, and all these compounds offer an affinity improve-ment of about 50-fold, with compound 21 being the best andmost ligand efficient. Figure 9a shows the crystal structure of21 and confirms the formation of a good hydrogen bondbetween the morpholine oxygen and Lys58. The seconddesign idea centered on trying to displace the side chain ofLys58 with larger tertiary amides that might then form goodlipophilic interactions with the protein. The lysine side chainitself forms no specific contacts with the enzyme and seemeda plausible candidate for displacement despite its relativelyclear positioning in the electron density. Compounds 24, 25,and 26 are representative molecules, and the isoindoline 24delivered an affinity improvement of several-hundred-fold.Figure 9b shows the crystal structure of 24 where it can beseen that the side chain of Lys58 has indeedmoved away andforms a salt bridge with Glu62. The space formerly occupiedby Lys58 accommodates the phenyl ring of the isoindolinewhich forms good lipophilic contact with Ala55, Lys58, andIle96. Interestingly and perhaps surprisingly, it was thereforepossible to increase potency significantly by either forming adirect hydrogen bond with the side chain of Lys58 ordisplacing the side chain with lipophilic groups. The bestamides from the previous synthesis iteration were thencombined with the isopropyl group. This yielded a further2- to 4-fold improvement for compounds 27, 28, and 29 (theerror in the ITC measurement is about 2-fold, so thisprobably represents a real improvement). The change tothe isopropyl group also reduced the lipophilicity by about0.5 unit.

The final change was to convert the best phenols intoresorcinols driven by the similarity of the binding mode ofour compounds to the natural product radicicol (Figure 5b).The morpholine compound 30 is 40-fold more potent thanthe corresponding 4-OH compound 27 and has an improvedcell activity of 140 nM. The isoindoline resorcinol 31 showssubnanomolar affinity, excellent ligand efficiency, and goodcell activity (31 nM). For compounds 30 and 31, the addi-tional hydroxyl group gave large improvements in affinity

Table 3. Optimization of the Phenol Series


but also gave much better translation between the measuredaffinities and the cell activity (for reasons that were notestablished). Figure 9c gives the binding mode of compound31 in Hsp90, and as anticipated, the additional hydroxylgroup forms good hydrogen bonds with Asp93 and a med-iating water molecule. The superimposition with the originalfragment demonstrates that the binding mode was preservedduring the fragment optimization campaign.

Figure 10 shows Western immunoblotting data forHCT116 cells treated with 31 and confirms that the com-pound has the anticipated molecular signature of an Hsp90inhibitor;3,10 the client protein CDK4 is depleted, while thechaperone protein Hsp70 is induced. These effects occur atconcentrations similar to the IC50 for the inhibition of cell

proliferation in the HCT116 cells. Compound 31was chosenas the lead compound, and further biological characteriza-tion of the series is described in the companion paper (DOI:10.1021/jm100060b).

Discussion and Conclusion

Wehave applied the Pyramid screening technique toHsp90and exploited a close integration of ligand observed NMRscreening and high throughput X-ray crystallography. Wefound it necessary to use a combination of cocrystallographyand soaking into different crystal forms of Hsp90 in orderto maximize the number of high quality X-ray structuresobtained. From this process, 26 fragment structures wereobtained during the screening phase of the project, and fourof these fragments are disclosed in this paper. Perhaps mostsurprising were fragments such as 4which bind to an inducedlipophilic pocket and the successful cosoaking of this frag-ment with 2-aminopyrimidine. Huth et al. have recentlydescribed a similar observation and detailed an NOE-basedNMR structure determination for the tertiary arrangement oftwo fragments, one of whichwas a simple aminopyrimidine.48

Choosing which fragments to work on is one of the parts offragment based discovery that requires judgment. Ligandefficiency39,40 is one criterion that we have used extensively,but it is not the only factor. Here fragment 3 had relativelypoor ligand efficiency (0.26) but its binding mode suggested a

Figure 9. Development of the lead compound from the phenol series. (a) Experimental binding mode of compound 21 illustrating theformation of a new hydrogen bondwith the side chain of Lys58. (b) Experimental bindingmode of compound 24 in which Lys58 is displaced bythe isoindoline group. In its new position, the side chain of Lys58 forms a salt bridge with Glu62. (c) Experimental binding mode of leadmolecule 31 (in cyan) superimposed on the original fragment 3 (in orange).

Figure 10. Effects of different concentrations of 31 on the levels ofCDK4 and Hsp70 in HCT116 cells as determined by Western blot.Included as controls are the levels of GAPDHprotein and the levelswhen no compound is added to HCT116 cells (shown as CTL).


number of design ideas for improvement, i.e., by convertingthe phenol into a resorcinol or by properly filling the proximallipophilic pocket. Experience on a variety of projects hasdemonstrated that ligand efficiency can be improved wherethere is a clear and correctable deficiency in the bindingmodeof the fragment.However, if this cannot be rapidly achieved, itis usually better to terminate work on such a fragment.Similarly, although fragment 1 had better ligand efficiency(0.38), its crystal structure suggested a design strategy toimprove potency, i.e., stabilizing the active conformationand filling the proximal lipophilic pocket. This emphasizesthat the binding mode of the fragment and the attractivenessof the associated medicinal chemistry plan are importantdeterminants in judging which fragments to pursue.

Subsequent to this work Brough et al. have disclosed aparallel virtual screening and fragment screening approach toHsp90.20 Aminopyrimidine based hits were identified by bothapproaches, and structure based drug design was used tohybridize hits from molecules with related binding modes.This approach successfully led to a clinical candidate. In ourwork, we have used virtual screening in a different manner toexplore SAR around very close analogues of identified frag-ments. For example, the bindingmode of fragment 3was usedto inform a virtual fragment screen culminating in the identi-fication of fragment 5. This more conservative approach mayallow more control over the molecular size and ligand effi-ciency of the developing lead series. Virtual screening ofdesigned compounds was also heavily used to prioritizesynthesis during fragment optimization.

Relatively small libraries of compounds are required toperform fragment screening, and if researchers rely on com-mercially available compounds, it is likely that the samefragments will appear in fragment screening collections fromdifferent companies. This represents an emerging challengefor FBDD practitioners to improve the novelty of fragmentsin their libraries and to establish strategies for introducingnovelty as molecules are optimized. Hsp90 is a good exampleon which to assess this because a large number of researchgroups have worked on this target and some of these groupshave used FBDD. The work on aminopyrimidines by Broughet al. illustrates how novelty can be introduced through thefusion of a heterocycle onto an initial aminopyrimidinefragment.20 Barker et al. identified aminopyrimidine frag-ments containing a semisaturated ring fused onto the amino-pyrimidine and presumably attained novelty as a result.49 Wefirst described our aminopyrimidine series in a patent severalyears ago,51 and subsequently Huth et al. have described thedevelopment of a similar series using FBDD.48 In the presentwork, the fragment screening and optimization approach aredifferent, and the final compound shows activity in cells.Resorcinol compounds have been worked on by a numberof groups.16 Both high throughput screening and fragmentscreening approaches were used in the development of NYP-AUY922 (Figure 2).21 The present work describes a purefragment-screening approach that originated from a frag-ment-like drug molecule containing a phenolamide moiety;the final compounds differ substantially in their architec-ture and shape from resorcinols such as NVP-AUY922which contain a five-membered aromatic heterocyle directlyattached to the resorcinol ring.22,52-56 Kung et al. havedescribed a resorcinol amide series that they identified inde-pendently via high throughput screening.57,58 The presentwork differs substantially in the screening and optimizationmethodology and in the final clinical candidate, which is

described in the companionpaper (DOI: 10.1021/jm100060b).Both our series59,60 and the series of Kung and co-workers61

have previously been described in the patent literature.The literature now contains many Hsp90 medicinal chem-

istry papers from a large number of groups. Approachesadopted include HTS, fragment screening, virtual screening,andnatural product optimizationoroptimizationbasedonanexisting lead.16-18 A number of clinical compounds have alsobeen revealed, so it is apparent that all approaches have beensuccessful to some degree and that Hsp90 is a reasonablytractable target fromamedicinal chemistrypoint of view.Thispaper shows that careful structure-based optimization basedon initially very weak fragment hits can deliver highly ligandefficient and low molecular weight leads for Hsp90 withoutthe need for large amounts of synthetic chemistry. In thecase of the pyrimidine series, optimization from fragment 1(250 μM) to compound 12 (6.3nM) improved affinity by40000-fold while the heavy atom count increased by just fiveatoms. This increase in affinitywasmainly gained through thestabilization of the active conformation of the starting frag-ment and the improvement of hydrophobic contact with theenzyme. Figure 11 and its caption summarize the structure-guided optimization process that led to the resorcinol leadcompound 31 (0.54 nM) starting from the phenol fragment 3(790 μM). Affinity was improved by over a million-fold withthe addition of only six heavy atoms, and this can be ascribedto three changes, each contributing about 100-fold to affinity.The first modification (methoxy to isopropyl) gave superiorfilling of the proximal lipophilic pocket with a net increase ofone heavy atom; the second modification (diethylamide toisoindolamide) was the least “efficient” with a net increase offour heavy atoms and was driven by improved lipophiliccontact with the enzyme as a result of the conformationalmovement of Lys58. The final modification (addition of ahydroxyl group) added one additional heavy atom with theformation of a hydrogen bond with Asp97 and a furtherwater-mediated hydrogen bond. In terms of the efficiency ofthe added groups,62 the two fragment to lead campaignsdescribed in this paper are among the most efficient everreported. In both cases the final lead has amolecularweight ofaround 300 Da which leaves a significant amount of “mole-cular weight head room” to tune the other properties of themolecule during lead optimization. This means that groups

Figure 11. Structure for lead molecule 31 together with fragment 3from which it was derived. The lead molecule is over 1 000 000 timesmore potent than the fragment, and this can be decomposed into(i) 110-fold for replacement of the methoxy group by isopropyl asderived from affinities for compound 3 and compound 17 (netincrease of one heavy atom), (ii) 100-fold for replacement of thediethylamide with the isoindoline amide as derived from affinitiesfor compound 17 and compound 28 (net increase of four heavyatoms), and (iii) 130-fold for the introduction of the 2-OH group asderived from affinities for compound 28 and compound 31 (netincrease of one heavy atom).


can be added to the molecule to improve nonpotency relatedpropertieswithout the fear that the resulting clinical candidatelies outside druglike space.

In conclusion we have applied a fragment screening ap-proach to Hsp90 and developed two series of compoundsstarting from fragments. In each series, structure-based drugdesign led to the discovery of high potency compounds withexcellent ligand efficiency. A resorcinol lead with subnano-molar affinity and good cell potency has been identified, andthe companion paper (DOI: 10.1021/jm100060b) describeshow this leadwas converted into a compound that is currentlyin clinical trials for the treatment of cancer.

Experimental Section

Chemistry. The method employed in determining the purityof compounds was LCMS, and all compounds had purities of>95%. Reagents and solvents were obtained from commercialsuppliers and used without further purification. Thin layerchromatography (TLC) analytical separations were conductedwith E. Merck silica gel F-254 plates of 0.25 mm thickness andwere visualized with UV light (254 nM) and/or stained withiodine, potassium permanganate, or phosphomolybdic acidsolutions followed by heating. Flash chromatography wasperformed either manually using E. Merck silica gel or usingan automated Biotage SP4 system with prepacked disposableSiO2 cartridges (4, 8, 19, 38, 40, and 90 g sizes) with stepped orgradient elution at 5-40 mL/min using the indicated solventmixture. 1H nuclear magnetic resonance (NMR) spectra wererecorded in the deuterated solvents specified on a BrukerAvance 400 spectrometer operating at 400 MHz. Chemicalshifts are reported in parts per million (δ) from the tetramethyl-silane resonance in the indicated solvent (TMS: 0.0 ppm). Dataare reported as follows: chemical shift, multiplicity (br=broad,s = singlet, d = doublet, t = triplet, m=multiplet), integration.Compound purity and mass spectra were determined by a Waters2795/Platform LC LC/MS system using the positive electrosprayionization technique (þESI), a Waters Fractionlynx/MicromassZQ LC/MS system using the positive electrospray ionizationtechnique (þESI), or anAgilent 1200SL-6140LC/MS systemusingpositive-negative switching, using a mobile phase of acetonitrile/water with 0.1% formic acid.

4-(3-Pyridyl)pyrimidin-2-ylamine 1, 2-aminopyrimidine 2, 4,N,N-diethyl-3-methoxy-4-hydroxybenzamide 3, 2-chloro-4-methyl-phthalazine 4, 4-chloro-(6-phenyl)pyrimidin-2-ylamine 5, 3-chloro-4-hydroxybenzoic acid, 3-tert-butyl-4-hydroxybenzoic acid, and5-tert-butyl-2-methoxybenzoic acid are commercially available.5-Isopropyl-2-methoxybenzoic acid,63 3-ethyl-4-hydroxybenzoicacid,64 and 2,4-bis-benzyloxy-5-isopropenylbenzoic acid59 wereprepared as described previously. 4-Hydroxy-3-isopropylben-zoic acid was prepared from 2-isopropylphenol according to themethod described64 for the synthesis of 3-ethyl-4-hydroxyben-zoic acid. Experimental and spectroscopic details for all othernoncommercially available compounds are given below or areavailable in the Supporting Information.

4-Chloro-6-[2,4-dichloro-5-(2-morpholin-4-ylethoxy)phenyl]-pyrimidin-2-ylamine (14). Concentrated HCl (19 mL) wasadded to 5-(benzyloxy)-2,4-dichloroaniline (52 g, 19.3 mmol,1.0 equiv) in glacial acetic acid (77 mL), and the mixture wascooled in an ice-water bath. Sodium nitrite (1.5 g, 22.2 mmol,1.2 equiv) was added in water (64 mL) slowly, maintaining thetemperature at <5 �C. This was stirred for 30 min. Thismixture was poured into KI (6.4 g, 38.6 mmol, 2.0 equiv) andI2 (1.7 g, 5.4 mmol, 0.3 equiv) in water (241 mL) and was left tostir for 1.5 h at room temperature. The reaction was quenchedby diluting with water and extracting the product with dichloro-methane (�3). The combined organic layers were washed withbrine and dried over MgSO4. The product was filtered andconcentrated to dryness under reduced pressure to leave

1-benzyloxy-2,4-dichloro-5-iodobenzene as a brown oil. Theproduct was taken on without further purification. To 1-ben-zyloxy-2,4-dichloro-5-iodobenzene (4.5 g, 11.9 mmol, 1.0equiv) in tetrahydrofuran (40 mL) at -78 �C was added thetriisopropyl borate (3.4 g, 17.8 mmol, 1.5 equiv) and then then-BuLi (11.1 mL, 1.6 M, 1.5 equiv) slowly. The mixture wasallowed to stir for an hour, and the reaction was then quenchedwith NH4Cl (sat., aq.). The mixture was allowed to warm toroom temperature and was diluted with water and ethylacetate. The product was extracted with ethyl acetate (�3).The combined organic layers were washed with brine and driedover MgSO4. The product was filtered and concentrated todryness under reduced pressure to leave a pale-yellow oil. Thecrude reaction mixture was coupled to 2-amino-4,6-dichloro-pyrimidine following the same procedure as for pyrimidine 7,except the reaction was heated thermally at 50 �C for 3 h. Thetitle compound was isolated by preparative HPLC to yield theproduct as a colorless solid. 1H NMR (400 MHz, Me-d3-OD)δ 7.60 (s, 1H), 7.49 (d, 2H), 7.45-7.32 (m, 4H), 6.93 (s, 1H),5.22 (s, 2H). LCMS (ESI): m/z 380 (M þ Hþ).

To 4-(5-benzyloxy-2,4-dichlorophenyl)-6-chloropyrimidin-2-ylamine (5.0 g, 9.2 mmol, 1.0 equiv) in dichloromethane(31 mL) was added BCl3 (10.1 mL, 10.1 mmol, 1.1 equiv) at-78 �C under a nitrogen atmosphere. The mixture was stirredfor 2 h. The reaction was then quenched with methanol (10mL),and themixture was allowed to warm to room temperature. Themixture was then diluted with water, and the product wasextracted with ethyl acetate (�3). The combined organic layerswere washed with water, brine and dried over MgSO4. Theproduct was filtered and concentrated to dryness under reducedpressure to leave a pale-yellow oil. The product was purified bycolumn chromatography (Rf = 0.48, 100% diethyl ether) toyield 5-(2-amino-6-chloropyrimidin-4-yl)-2,4-dichlorophenolas a colorless solid. 1H NMR (400 MHz, Me-d3-OD) δ 7.48 (s,1H), 7.11 (s, 1H), 6.90 (s, 1H). LCMS (ESI):m/z 290 (MþHþ).

To 5-(2-amino-6-chloropyrimidin-4-yl)-2,4-dichlorophenol(81 mg, 0.28 mmol, 1.0 equiv) in DMF (3 mL) was addedCs2CO3 (199 mg, 0.61 mmol, 2.1 equiv) and then N-(2-chloro-ethyl)morpholine 3HCl (57 mg, 0.31 mmol, 1.1 equiv) under anitrogen atmosphere. Themixturewas heated thermally at 80 �Cfor 3 h. The mixture was allowed to cool and concentrated todryness under reduced pressure. The title compound 14 wascrystallized frommethanol to yield the product as beige crystals.1HNMR(400MHz,Me-d3-OD)δ 7.58 (s, 1H), 7.33 (s, 1H), 6.96(s, 1H), 4.27 (t, 2H), 3.72 (dd, 4H), 2.88 (t, 2H), 2.66 (dd, 4H).LCMS (ESI): m/z 403 (M þ Hþ). LCMS purity: 96.7%.

4-(2,4-Dihydroxy-5-isopropylbenzoyl)morpholine (30).Morpho-line (66 mg, 0.75 mmol) was added to a stirred mixture of 2,4-bis-benzyloxy-5-isopropenylbenzoic acid (187 mg, 0.5 mmol), 1-(3-di-methylaminopropyl)-3-ethylcarbodimide hydrochloride (116 mg,1.2 mmol), and 1-hydroxybenzotriazole (81 mg, 0.6 mmol) indichloromethane (10 mL), and the mixture was stirred at roomtemperature for 4h.Themixturewaswashed successivelywith 2Mhydrochloric acid, saturated sodium bicarbonate, and water. Theorganic layer was separated and concentrated under reducedpressure to afford 4-(2,4-bis-benzyloxy-5-isopropenylbenzoyl)-morpholine as a colorless solid. Yield: 76%. 1H NMR (DMSO-d6) δ 7.57-7.30 (m, 10H), 7.02 (s, 1H), 6.95 (s, 1H), 5.18 (d, 4H),5.06 (d, 2H), 3.57 (br s, 4H), 3.41 (br s, 2H), 3.16 (br s, 2H), 2.03 (s,3H). LCMS (þESI): m/z 444 (M þ Hþ). LCMS purity: 95.8%.

4-(2,4-Bis-benzyloxy-5-isopropenylbenzoyl)morpholine (133mg, 0.3 mmol) was dissolved in methanol (8 mL), 10% palla-dium on carbon (50 mg) was added, and the mixture was stirredovernight under a hydrogen atmosphere at room temperature.The mixture was filtered, the solids were rinsed with methanol(4mL), and the combined fitrates were reduced to dryness underreduced pressure to afford 30 as a colorless solid.Yield: 90%. 1HNMR (DMSO-d6): δ 9.57 (br s, 1H), 6.85 (s, 1H), 6.36 (s, 1H),3.56 (br m, 4H), 3.40 (br s, 4H), 3.06 (m, 1H), 1.10 (d, 6H).LCMS (þESI): m/z 266 (M þ Hþ). LCMS purity: 97.1%.


2-(2,4-Dihydroxy-5-isopropylbenzoyl)-2,3-dihydro-1H-isoindole(31). Compound 31 was prepared in a similar manner to 30 from2,3-dihydro-1H-isoindole and 2,4-bis-benzyloxy-5-isopropenyl-benzoic acid. 1H NMR (DMSO-d6): δ 10.04 (s, 1H), 9.64 (s,1H), 7.31 (brm, 4H, s), 7.04 (s, 1H), 6.41 (s, 1H), 4.77 (s, 4H), 3.10(m, 1H), 1.14 (d, 6H, d). LCMS (þESI): m/z 298 (M þ Hþ).LCMS purity: 100.0%.

NMR Screening. NMR experiments were carried out at500 MHz, using a Bruker DRX500 instrument equipped with aTXIcryoprobe. TheN-terminal domain ofHsp90was expressedand purified as described below, giving a product of molecularmass 24 508 Da, whose mass was confirmed by time-of-flightmass spectrometry. Samples for NMR screening were preparedin 20 mMTris buffer, pH7.4, containing 200 μMADP, 150 mMNaCl, 1 mM BME, and 10% D2O. Typical screening samplescontained 0.3mg/mL (12.5 μM)Hsp90 and four fragments, eachat 500 μM (2%DMSO). This gave a molar ratio of fragment totarget of 40:1, a typical value for water-LOGSY experiments.44

At these protein and ligand concentrations, fragments that bindwith dissociation constants better than 1 mM are expected to bedetected. LOGSY experiments were performed using thee-PHOGSY sequence of Dalvit et al. incorporating a 1 s delayto allow cross-relaxation between fragments and water, and a100 ms CPMG period during which time any protein magneti-zation decays.65

In order to enhance the LOGSY signal, all NMR experimentswere performed at 5 �C. Lowering the temperature improves theaffinities of ligands that bind exothermically and reduces therates of exchange between bulk water and water of hydration,leading to improved LOGSY intensities. In order to detect thesmall positive LOGSY effects caused by binding to the proteintarget, LOGSY spectra were obtained in the presence and theabsence of the protein target, and these spectra were directlycompared.

The LOGSY spectra were defined as medium or strongsignals as follows. LOGSY spectra were obtained from twoseparate samples (þprotein and buffer control), and these weresubtracted (þprotein - buffer control) to give the LOGSYdifference spectrum. This spectrum shows the change in theamount of magnetization transferred from water to the ligands,due to protein binding. The intensity of each peak in thisspectrum depends on a number of experimental choices(number of scans, magnetization transfer time, relaxation de-lays, etc.), and there is no reference spectrum generated withwhich to quantitate the LOGSY effects. However a standard 1Dspectrumwas also run (with fewer scans and different relaxationdelays) and the LOGSY intensity was compared with this. Foreach compound, the largest %LOGSY signal observed for anydetectable aromatic proton was recorded. The %LOGSY va-lues for ligands against HSP90 (LOGSY intensity versus 1Dintensity) ranged from 0 to about 60% with strong LOGSYsignals being defined as >20% and medium signals defined as11-20%.

The presence of 200 μM ADP in the buffer enabled fragmenthits, which had been identified by changes in their LOGSYsignals, to be further characterized according to their competitionat the nucleotide site, determined by a reduction in the LOGSYsignal of ADP. In the absence ofMg2þ, 200 μMcorresponds to aconcentrationofADParound its dissociation constant so that thepresence of ADP does not greatly reduce the sensitivity of theexperiment to fragment binding while providing a convenientmethod of identifying cocktails that contain an active-site ligand.LOGSY spectra were reacquired after the addition of a smallaliquot of a concentrated MgCl2 solution to the screening solu-tions, giving a final concentration of 5mM.This convertsADP toits Mg2þ complex which binds more tightly to Hsp90, with adissociation constant measured by ITC of approximately 10 μM.This in turn leads to complete displacement of fragments thatbind exclusively to the active site of Hsp90, enabling selectiveactive-site binders to be readily identified.

Crystallography. Two hexahistidine tagged N-terminal frag-ments (9-236 and 9-224) of Hsp90R were cloned into a pET28vector and expressed in BL21 (DE3). The proteins were cap-tured using a Ni2þ affinity column followed by removal of thetag using thrombin and final purification on a Superdex 75 gelfiltration column with a final buffer of 20 mM Tris-HCl (pH7.4), 150 mM NaCl, and 1 mM β-ME. The proteins wereconcentrated to between 20 and 25 mg mL-1 prior to crystal-lization. For soaking experiments, two crystal forms wereexploited because of the differing loop structure around theactive site. The I222 apo crystals were generated using thehanging drop, vapor diffusion method by combining equalvolumes of protein and mother liquor (0.1 M Hepes 3NaOH,pH 7.2, 0.2 M magnesium chloride, 20% (w/v) MPEG 2000).Structures for compounds 2, 4, 2 þ 4, and 14 were obtained inthis system. The P21 back-soakable crystals were also generatedusing the hanging drop, vapor diffusion method but 2 mMADP 3Mg2þ was added to the protein before combining withmother liquor (0.1 M Bis-Tris, pH 6.0, 0.2 M ammoniumphosphate, 25% (w/v) PEG 3350). Structures for compounds6, 18, and 21 were obtained in this system. All crystallizationexperiments were performed at 4 �C and usable crystalsappeared between 1 and 3 days. Soaking was performed inrespective mother liquors containing 20% glycerol and 50 mMcompound with a final DMSO concentration of 10% (v/v) forperiods of up to 24 h. Crystals were subsequently flash frozenand data collected on a Rigaku RU-H3R rotating anode gen-erator withOsmic confocal blue optics and either a Jupiter CCDorRaxis 4þþ image plate. For cocrystallization, the proteinwasmixed with 5 mM compound with a final DMSO concentrationof 2.5% (v/v), followed by automated crystallization setup usinga Cartesian robot, typically at 0.2 μL final drop volumes insitting drop 96-well formats using a limited in house designedmatrix of crystallization conditions. Crystals grown using thissystem were extracted from the low volume drops, cryopro-tected in mother liquor with 20% glycerol, and flash frozen forin house data collection. Compound 1 crystals were grown in0.1 M Bis-Tris, pH 5.5, 0.2 M ammonium acetate, 25% PEG3350. Compound 3 crystals were grown in 0.1 M Bis-Tris,pH 5.75, 0.2Mammonium acetate, 20%PEG3350. Compound24 crystals were grown in 0.1 M Bis-Tris, pH 5.25, 0.2 Mammonium acetate, 27% PEG 3350. Compound 31 crystalswere grown in 0.1 M Tris-HCl, pH 8.0, 0.2 M magnesiumchloride, 22% PEG 4000.

Diffractiondatawere processed usingMOSFLM66 andCCP4.67

The structureswere solvedusing isomorphous replacementwith thecoordinates previously described, 1YER68 for the I222 form and1BYQ69 for theP21 back-soakable and cocrystallization form. Thestructures were refined using REFMAC570 and Buster-TNT.71

Ligands were placed using AutoSolve72 andmanual rebuilds madein AstexViewer273 and COOT.74 Final validation checks wereperformed using Procheck,75 prior to deposition of coordinatesand structure factors into the PDB.

Isothermal Titration Calorimetry (ITC). ITC experimentswere performed on a MicroCal VP-ITC at 25 �C in a buffercomprising 25 mM Tris, 100 mM NaCl, 1 mM MgCl2, and1 mM TCEP at pH 7.4. The final DMSO concentration wasbetween 1% and 5%. The protein used was the same Hsp90N-terminal ATPase domain construct used in both the NMRandX-ray crystallographywork.Most of ITC experiments wereset up with protein in the sample cell and compound in theinjection syringe, although in cases where compound solubilitywas limiting, this was reversed. Data were fit to a single sitebinding model using Origin 7.0 software. All the stoichiometryvalues from the data analysis were in the range 0.8-1.3,providing an excellent internal control for the quality, purity,and stability of both the protein and the compounds. Replicateexperiments indicate that errors in the dissociation constants aregenerally better than 2-fold. The stoichiometry parameter wasfixed at 1 in caseswhere theKd valuewas greater than the protein


concentration.76 By use of the procedure outlined above, ADPand 17-DMAG had measured dissociation constants of 9.2 and0.21 μM. These values are in good agreement with literaturedissociation constants with the full length humanHsp90 proteinof 11 μM for ADP and 0.35 μM for 17-DMAG.77 These resultsindicate that ITC on N-terminal Hsp90 is a good surrogate forITC on full length Hsp90, provided the compounds bind in theATPase active site. A competition format ITC78 was necessaryto accurately determine the affinity of compound 31. Thisrequired preincubating the protein with one of our moderatelypotent phenol compounds in the sample cell prior to initiatingthe titration with compound 31. A competition binding modelwas used to fit the data and obtain a Kd estimate for compound31. More experimental detail on ITC is given in SupportingInformation together with representative data for three com-pounds of different affinity (i.e., compounds 1, 5, and 31).

Calculation of the Torsion Energy Profile. All energies werecalculated using the ab initio quantum chemistry packageQ-Chem.79 To calculate the torsion energy profile, a torsionscan was carried out on a Corina generated structure by varyingthe desired torsion angle between 0� and 180�, in 15� intervals.For each structure at a given torsion angle, a constrained energyminimization (basis set, 3-21G*; method, Hartree-Fock) wascarried out with a single constraint on the torsion angle torestrain themolecule to the specific torsion angle value while therest of the molecule was geometrically optimized. The energy ofthe optimized molecule at the specified conformational torsionwas then calculated using B3LYP method with 6-311G* basisset. Finally, the 12 energies were utilized to estimate approxi-mately the relative population of each conformation using aBoltzman distribution.

Virtual Screening. The X-ray crystal structure of the N-term-inal domain of Hsp90 complexed with compound 1 was used tovirtual-screen aminopyrimidine analogues of 1. Water mole-cules were removed from the structure with the exception of thefour conserved waters at the bottom of the ATP binding pocket.Hydrogens were added to the protein and waters, and a bindingpocket was generated with all protein and water atoms within6 A of any non-hydrogen atom in ligand 1. This binding site wasused to run all dockings and virtual screens in the presence of thekey water molecules, allowing them to toggle on/off and spinaround their three principal axes with methods and settingspreviously described by Verdonk et al.80 All calculations wererun on aLinux cluster using theAstexWeb-based virtual screen-ing platform.81 A number of filters (heavy atom count, mole-cular weight, number of donors, number of acceptors, ClogP,and polar surface area) combined with the scoring functionsGoldscore82 and Chemscore83,84 were applied to score and torank the docked libraries. Of the two scoring functions, Gold-score performed generally better in terms of reproducing thecorrect binding modes observed in Hsp90 X-ray crystal struc-tures. Finally, virtual screens were visually analyzed, and mole-cules were selected on the basis of the binding mode, thechemical tractability, and the calculated score.

Hsp90 Cell Assay and Western Blot. The antiproliferativeactivities of the compounds were determined bymeasuring theirability to inhibit the growth of HCT116 cells (obtained fromEuropean Collection of Cell Cultures). Cell growth was mea-sured using theAlamar blue assay as described in Squires et al.85

ForWestern blotting experiments, HCT116 cells were treatedwith compound in six-well tissue culture plates at the statedconcentrations for 18 h before washing with PBS and lysing cellsby the addition of ice-cold lysis buffer (40 mM Tris-HCl (pH7.5), 274 mM NaCl, 20% glycerol, 2% Triton-X-100, 1 mMNa3VO4, 50 mM NaF, 1% protease inhibitor cocktail set III(Calbiochem)). The cells were lysed on ice for 30 min andclarified by centrifugation. Lysates in SDS sample buffer wereheated for 5 min at 95 �C, resolved by SDS-PAGE, andimmunoblotted with the indicated antibodies followed byeither infrared dye labeled anti-rabbit or anti-mouse antibodies

(LICOR). Antibodies were sourced as follows: CDK4 fromSantaCruzAntibodies (SantaCruzCA),Hsp70 fromStressGenBiotechnologies (Victoria, British Columbia, Canada), andGAPDH from Chemicon International (Temecula, CA). Blotswere scanned to detect infrared fluorescence on the Odysseyinfrared imaging system (LICOR).

Acknowledgment. The authors thankAnneCleasby,DavidRees, Brian Williams, Lyn Leaper, Harren Jhoti, Robin Carr,Neil Thompson, and John Lyons for comments on themanuscript and for providing support to the project.

Supporting Information Available: Additional syntheticchemistry experimental information, additional ITC experi-mental information, and representative examples of ITC data.Thismaterial is available free of charge via the Internet at http://pubs.acs.org.

References

(1) Bukau, B.; Weissman, J.; Horwich, A. Molecular chaperones andprotein quality control. Cell 2006, 125, 443–451.

(2) Workman, P.; Burrows, F.; Neckers, L.; Rosen, N. Drugging thecancer chaperone HSP90: combinatorial therapeutic exploitationof oncogene addiction and tumor stress.Ann. N.Y. Acad. Sci. 2007,1113, 202–216.

(3) Whitesell, L.; Lindquist, S. L. HSP90 and the chaperoning ofcancer. Nat. Rev. Cancer 2005, 5, 761–772.

(4) Hanahan, D.; Weinberg, R. A. The hallmarks of cancer.Cell 2000,100, 57–70.

(5) Workman, P. Combinatorial attack on multistep oncogenesis byinhibiting the Hsp90 molecular chaperone. Cancer Lett. 2004, 206,149–157.

(6) Chiosis, G.; Neckers, L. Tumor selectivity of Hsp90 inhibitors:the explanation remains elusive. ACS Chem. Biol. 2006, 1, 279–284.

(7) Kamal, A.; Thao, L.; Sensintaffar, J.; Zhang, L.; Boehm, M. F.;Fritz, L. C.; Burrows, F. J. A high-affinity conformation of Hsp90confers tumour selectivity on Hsp90 inhibitors. Nature 2003, 425,407–410.

(8) Solit, D. B.; Chiosis, G. Development and application of Hsp90inhibitors. Drug Discovery Today 2008, 13, 38–43.

(9) Pearl, L.H.; Prodromou,C. Structure andmechanismof theHsp90molecular chaperone machinery. Annu. Rev. Biochem. 2006, 75,271–294.

(10) Vilenchik, M.; Solit, D.; Basso, A.; Huezo, H.; Lucas, B.; He, H.;Rosen, N.; Spampinato, C.; Modrich, P.; Chiosis, G. Targetingwide-range oncogenic transformation via PU24FCl, a specificinhibitor of tumor Hsp90. Chem. Biol. 2004, 11, 787–797.

(11) Soga, S.; Shiotsu, Y.; Akinaga, S.; Sharma, S. V. Development ofradicicol analogues. Curr. Cancer Drug Targets 2003, 3, 359–369.

(12) Neckers, L.; Schulte, T. W.; Mimnaugh, E. Geldanamycin as apotential anti-cancer agent: its molecular target and biochemicalactivity. Invest. New Drugs 1999, 17, 361–373.

(13) Ge, J.; Normant, E.; Porter, J. R.; Ali, J. A.; Dembski, M. S.; Gao,Y.; Georges, A. T.; Grenier, L.; Pak, R. H.; Patterson, J.; Sydor,J. R.; Tibbitts, T. T.; Tong, J. K.; Adams, J.; Palombella, V. J.Design, synthesis, and biological evaluation of hydroquinonederivatives of 17-amino-17-demethoxygeldanamycin as potent,water-soluble inhibitors of Hsp90. J. Med. Chem. 2006, 49, 4606–4615.

(14) Kaur, G.; Belotti, D.; Burger, A. M.; Fisher-Nielson, K.; Borsotti,P.; Riccardi, E.; Thillainathan, J.; Hollingshead, M.; Sausville,E. A.; Giavazzi, R. Antiangiogenic properties of 17-(dimethyl-aminoethylamino)-17-demethoxygeldanamycin: an orally bioa-vailable heat shock protein 90 modulator. Clin. Cancer Res. 2004,10, 4813–4821.

(15) Schulte, T. W.; Neckers, L. M. The benzoquinone ansamycin 17-allylamino-17-demethoxygeldanamycin binds to HSP90 andshares important biologic activities with geldanamycin. CancerChemother. Pharmacol. 1998, 42, 273–279.

(16) Drysdale, M. J.; Brough, P. A. Medicinal chemistry of Hsp90inhibitors. Curr. Top. Med. Chem 2008, 8, 859–868.

(17) Janin, Y. L. Heat shock protein 90 inhibitors. A text book exampleof medicinal chemistry? J. Med. Chem. 2005, 48, 7503–7512.

(18) Blagg, B. S.; Kerr, T. D. Hsp90 inhibitors: small molecules thattransform the Hsp90 protein folding machinery into a catalyst forprotein degradation. Med. Res. Rev. 2006, 26, 310–338.


(19) Sgobba, M.; Rastelli, G. Structure-based and in silico design ofHsp90 inhibitors. ChemMedChem 2009, 4, 1399–1409.

(20) Brough, P. A.; Barril, X.; Borgognoni, J.; Chene, P.; Davies,N. G.; Davis, B.; Drysdale, M. J.; Dymock, B.; Eccles, S. A.;Garcia-Echeverria, C.; Fromont, C.; Hayes, A.; Hubbard, R. E.;Jordan, A. M.; Jensen, M. R.; Massey, A.; Merrett, A.; Padfield,A.; Parsons, R.; Radimerski, T.; Raynaud, F. I.; Robertson,A.; Roughley, S. D.; Schoepfer, J.; Simmonite, H.; Sharp,S. Y.; Surgenor, A.; Valenti, M.; Walls, S.; Webb, P.; Wood, M.;Workman, P.; Wright, L. Combining hit identification strategies:fragment-based and in silico approaches to orally active 2-amino-thieno[2,3-d]pyrimidine inhibitors of the Hsp90 molecular chaper-one. J. Med. Chem. 2009, 52, 4794–4809.

(21) Eccles, S. A.; Massey, A.; Raynaud, F. I.; Sharp, S. Y.; Box, G.;Valenti, M.; Patterson, L.; de Haven, B. A.; Gowan, S.; Boxall, F.;Aherne, W.; Rowlands, M.; Hayes, A.; Martins, V.; Urban, F.;Boxall, K.; Prodromou, C.; Pearl, L.; James, K.; Matthews, T. P.;Cheung, K. M.; Kalusa, A.; Jones, K.; McDonald, E.; Barril, X.;Brough, P. A.; Cansfield, J. E.; Dymock, B.; Drysdale, M. J.;Finch, H.; Howes, R.; Hubbard, R. E.; Surgenor, A.; Webb, P.;Wood, M.; Wright, L.; Workman, P. NVP-AUY922: a novelheat shock protein 90 inhibitor active against xenograft tumorgrowth, angiogenesis, and metastasis. Cancer Res. 2008, 68, 2850–2860.

(22) Brough, P. A.; Aherne, W.; Barril, X.; Borgognoni, J.; Boxall, K.;Cansfield, J. E.; Cheung, K. M.; Collins, I.; Davies, N. G.; Drys-dale, M. J.; Dymock, B.; Eccles, S. A.; Finch, H.; Fink, A.; Hayes,A.; Howes, R.; Hubbard, R. E.; James, K.; Jordan, A. M.; Lockie,A.; Martins, V.; Massey, A.; Matthews, T. P.; McDonald, E.;Northfield, C. J.; Pearl, L. H.; Prodromou, C.; Ray, S.; Raynaud,F. I.; Roughley, S. D.; Sharp, S. Y.; Surgenor, A.;Walmsley, D. L.;Webb, P.; Wood, M.; Workman, P.; Wright, L. 4,5-Diarylisox-azole Hsp90 chaperone inhibitors: potential therapeutic agents forthe treatment of cancer. J. Med. Chem. 2008, 51, 196–218.

(23) Bao, R.; Lai, C. J.; Qu, H.; Wang, D.; Yin, L.; Zifcak, B.; Atoyan,R.;Wang, J.; Samson,M.; Forrester, J.; DellaRocca, S.; Xu, G. X.;Tao, X.; Zhai, H. X.; Cai, X.; Qian, C. CUDC-305, a novelsynthetic HSP90 inhibitor with unique pharmacologic propertiesfor cancer therapy. Clin. Cancer Res. 2009, 15, 4046–4057.

(24) Huang, K. H.; Veal, J. M.; Fadden, R. P.; Rice, J. W.; Eaves, J.;Strachan, J. P.; Barabasz, A. F.; Foley, B. E.; Barta, T. E.;Ma,W.;Silinski, M. A.; Hu, M.; Partridge, J. M.; Scott, A.; DuBois, L. G.;Freed, T.; Steed, P.M.; Ommen, A. J.; Smith, E. D.; Hughes, P. F.;Woodward, A. R.; Hanson, G. J.; McCall, W. S.; Markworth,C. J.; Hinkley, L.; Jenks, M.; Geng, L.; Lewis, M.; Otto, J.; Pronk,B.; Verleysen,K.;Hall, S. E.Discovery of novel 2-aminobenzamideinhibitors of heat shock protein 90 as potent, selective and orallyactive antitumor agents. J. Med. Chem. 2009, 52, 4288–4305.

(25) Lundgren, K.; Zhang, H.; Brekken, J.; Huser, N.; Powell, R. E.;Timple, N.; Busch,D. J.; Neely, L.; Sensintaffar, J. L.; Yang, Y. C.;McKenzie, A.; Friedman, J.; Scannevin, R.; Kamal, A.; Hong, K.;Kasibhatla, S. R.; Boehm,M. F.; Burrows, F. J. BIIB021, an orallyavailable, fully synthetic small-molecule inhibitor of the heat shockprotein Hsp90. Mol. Cancer Ther. 2009, 8, 921–929.

(26) Erlanson, D. A.; McDowell, R. S.; O’Brien, T. Fragment-baseddrug discovery. J. Med. Chem. 2004, 47, 3463–3482.

(27) Rees, D. C.; Congreve, M.; Murray, C. W.; Carr, R. Fragment-based lead discovery. Nat. Rev. Drug Discovery 2004, 3, 660–672.

(28) Erlanson, D. A. Fragment-based lead discovery: a chemical up-date. Curr. Opin. Biotechnol. 2006, 17, 643–652.

(29) Fattori, D. Molecular recognition: the fragment approach in leadgeneration. Drug Discovery Today 2004, 9, 229–238.

(30) Hajduk, P. J.; Greer, J. A decade of fragment-based drug design:strategic advances and lessons learned. Nat. Rev. Drug Discovery2007, 6, 211–219.

(31) Alex, A. A.; Flocco, M. M. Fragment-based drug discovery: whathas it achieved so far? Curr. Top. Med. Chem. 2007, 7, 1544–1567.

(32) Congreve, M.; Chessari, G.; Tisi, D.; Woodhead, A. J. Recentdevelopments in fragment-based drug discovery. J. Med. Chem.2008, 51, 3661–3680.

(33) Chessari,G.;Woodhead,A. J.Fromfragment to clinical candidate;ahistorical perspective. Drug Discovery Today 2009, 14, 668–675.

(34) Schulz, M. N.; Hubbard, R. E. Recent progress in fragment-basedlead discovery. Curr. Opin. Pharmacol. 2009, 9, 615–621.

(35) Leeson, P.D.; Springthorpe, B. The influence of drug-like conceptson decision-making in medicinal chemistry. Nat. Rev. Drug Dis-covery 2007, 6, 881–890.

(36) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J.Experimental and computational approaches to estimate solubilityand permeability in drug discovery and development settings.Adv.Drug Delivery Rev. 2001, 46, 3–26.

(37) Vieth, M.; Siegel, M. G.; Higgs, R. E.; Watson, I. A.; Robertson,D. H.; Savin, K. A.; Durst, G. L.; Hipskind, P. A. Characteristicphysical properties and structural fragments of marketed oraldrugs. J. Med. Chem. 2004, 47, 224–232.

(38) Wenlock, M. C.; Austin, R. P.; Barton, P.; Davis, A. M.; Leeson,P. D. A comparison of physiochemical property profiles of devel-opment and marketed oral drugs. J. Med. Chem. 2003, 46, 1250–1256.

(39) Hopkins, A. L.; Groom, C. R.; Alex, A. Ligand efficiency: a usefulmetric for lead selection. Drug Discovery Today 2004, 9, 430–431.

(40) Kuntz, I. D.; Chen, K.; Sharp, K. A.; Kollman, P. A. The maximalaffinity of ligands. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 9997–10002.

(41) Leach,A.R.;Hann,M.M.;Burrows, J.N.;Griffen, E. J. Fragmentscreening: an introduction. Mol. BioSyst. 2006, 2, 430–446.

(42) Hann,M.M.; Leach, A. R.; Harper, G.Molecular complexity andits impact on the probability of finding leads for drug discovery.J. Chem. Inf. Comput. Sci. 2001, 41, 856–864.

(43) Hartshorn, M. J.; Murray, C. W.; Cleasby, A.; Frederickson, M.;Tickle, I. J.; Jhoti, H. Fragment-based lead discovery using X-raycrystallography. J. Med. Chem. 2005, 48, 403–413.

(44) Dalvit, C.; Fogliatto, G.; Stewart, A.; Veronesi, M.; Stockman, B.WaterLOGSY as a method for primary NMR screening: practicalaspects and range of applicability. J Biomol. NMR 2001, 21, 349–359.

(45) Wolfson, B.; Siker, E. S.; Ciccarelli, H. E. A double blind compar-ison of doxapram, ethamivan and methylphenidate. Am. J. Med.Sci. 1965, 249, 391–398.

(46) Roe, S. M.; Prodromou, C.; O’Brien, R.; Ladbury, J. E.; Piper,P. W.; Pearl, L. H. Structural basis for inhibition of the Hsp90molecular chaperone by the antitumor antibiotics radicicol andgeldanamycin. J. Med. Chem. 1999, 42, 260–266.

(47) Wright, L.; Barril, X.; Dymock, B.; Sheridan, L.; Surgenor, A.;Beswick, M.; Drysdale, M.; Collier, A.; Massey, A.; Davies,N.; Fink, A.; Fromont, C.; Aherne, W.; Boxall, K.; Sharp, S.;Workman, P.; Hubbard, R. E. Structure-activity relationships inpurine-based inhibitor binding to HSP90 isoforms. Chem. Biol.2004, 11, 775–785.

(48) Huth, J. R.; Park, C.; Petros, A.M.; Kunzer, A. R.; Wendt,M. D.;Wang, X.; Lynch, C. L.; Mack, J. C.; Swift, K. M.; Judge, R. A.;Chen, J.; Richardson, P. L.; Jin, S.; Tahir, S. K.; Matayoshi, E. D.;Dorwin, S. A.; Ladror,U. S.; Severin, J.M.;Walter, K.A.; Bartley,D. M.; Fesik, S. W.; Elmore, S. W.; Hajduk, P. J. Discovery anddesign of novel HSP90 inhibitors using multiple fragment-baseddesign strategies. Chem. Biol. Drug Des. 2007, 70, 1–12.

(49) Barker, J. J.; Barker, O.; Boggio, R.; Chauhan, V.; Cheng, R. K.;Corden, V.; Courtney, S. M.; Edwards, N.; Falque, V. M.; Fusar,F.; Gardiner, M.; Hamelin, E. M.; Hesterkamp, T.; Ichihara, O.;Jones, R. S.; Mather, O.; Mercurio, C.; Minucci, S.; Montalbetti,C. A.; Muller, A.; Patel, D.; Phillips, B. G.; Varasi, M.; Whittaker,M.; Winkler, D.; Yarnold, C. J. Fragment-based identification ofHsp90 inhibitors. ChemMedChem 2009, 4, 963–966.

(50) Allen, F. H. The Cambridge Structural Database: a quarter of amillion crystal structures and rising. Acta Crystallogr. B 2002, 58,380–388.

(51) Chessari, G.; Congreve, M.; Callaghan, O.; Cowan, S.; Murray,C.W.; Woolford, A. J.; O’Brien, M. A.; Woodhead, A. J. Prepara-tion of Azinamines as Modulators of Heat Shock Protein 90(Hsp90). WO2006123165, 2006.

(52) Feldman,R. I.;Mintzer, B.; Zhu,D.;Wu, J.M.; Biroc, S. L.; Yuan,S.; Emayan,K.; Chang, Z.; Chen,D.; Arnaiz,D.O.; Bryant, J.; Ge,X. S.; Whitlow,M.; Adler, M.; Polokoff, M. A.; Li, W.W.; Ferrer,M.; Sato, T.; Gu, J.M.; Shen, J.; Tseng, J. L.; Dinter,H.; Buckman,B. Potent triazolothione inhibitor of heat-shock protein-90.Chem.Biol. Drug Des. 2009, 74, 43–50.

(53) Cikotiene, I.; Kazlauskas, E.; Matuliene, J.; Michailoviene, V.;Torresan, J.; Jachno, J.; Matulis, D. 5-Aryl-4-(5-substituted-2,4-dihydroxyphenyl)-1,2,3-thiadiazoles as inhibitors of Hsp90 chape-rone. Bioorg. Med. Chem. Lett. 2009, 19, 1089–1092.

(54) Cheung, K. M.; Matthews, T. P.; James, K.; Rowlands, M. G.;Boxall, K. J.; Sharp, S. Y.; Maloney, A.; Roe, S. M.; Prodromou,C.; Pearl, L. H.; Aherne, G. W.; McDonald, E.; Workman, P.The identification, synthesis, protein crystal structure and invitro biochemical evaluation of a new 3,4-diarylpyrazole classof Hsp90 inhibitors. Bioorg. Med. Chem. Lett. 2005, 15, 3338–3343.

(55) Dymock, B.W.; Barril, X.; Brough, P. A.; Cansfield, J. E.;Massey,A.;McDonald, E.; Hubbard, R. E.; Surgenor, A.; Roughley, S. D.;Webb, P.;Workman, P.; Wright, L.; Drysdale,M. J. Novel, potentsmall-molecule inhibitors of the molecular chaperone Hsp90 dis-covered through structure-based design. J. Med. Chem. 2005, 48,4212–4215.


(56) Gopalsamy, A.; Shi, M.; Golas, J.; Vogan, E.; Jacob, J.; Johnson,M.; Lee, F.; Nilakantan,R.; Petersen,R.; Svenson,K.; Chopra,R.;Tam, M. S.; Wen, Y.; Ellingboe, J.; Arndt, K.; Boschelli, F.Discovery of benzisoxazoles as potent inhibitors of chaperone heatshock protein 90. J. Med. Chem. 2008, 51, 373–375.

(57) Kung, P. P.; Funk, L.; Meng, J.; Collins, M.; Zhou, J. Z.; Johnson,M. C.; Ekker, A.; Wang, J.; Mehta, P.; Yin, M. J.; Rodgers, C.;Davies, J. F.; Bayman, E.; Smeal, T.; Maegley, K. A.; Gehring,M. R. Dihydroxylphenyl amides as inhibitors of the Hsp90molecular chaperone. Bioorg. Med. Chem. Lett. 2008, 18, 6273–6278.

(58) Kung, P. P.; Huang, B.; Zhang, G.; Zhou, J. Z.; Wang, J.; Digits,J. A.; Skaptason, J.; Yamazaki, S.; Neul, D.; Zientek,M.; Elleraas,J.; Mehta, P.; Yin, M. J.; Hickey, M. J.; Gajiwala, K. S.; Rodgers,C.; Davies, J. F.; Gehring, M. R. Dihydroxyphenylisoindolineamides as orally bioavailable inhibitors of the heat shock protein90 (Hsp90)molecular chaperone. J.Med. Chem. 2010, 53, 499–503.

(59) Chessari, G.; Congreve, M.; Figueroa, E.; Frederickson, M.;Murray, C. W.; Woolford, A. J.; Carr, M. G.; O’Brien, M. A.;Woodhead, A. J. Preparation of Benzamides as Hsp90 Inhibitors.WO2006109075, 2006.

(60) Chessari, G.; Congreve, M.; Figueroa, E.; Frederickson, M.;Murray, C. W.; Woolford, A. J.; Carr, M. G.; Downham, R.;O’Brien, M. A.; Phillips, T. R.; Woodhead, A. J. Preparationof Hydroxybenzamides as Hsp90 Inhibitors. WO2006109085,2006.

(61) Funk, L. A.; Johnson, M. C.; Kung, P. P.; Meng, J. J.; Zhou, J. Z.Preparation of Hydroxyarylcarboxamide Derivatives for TreatingCancer. WO2006117669, 2006.

(62) Verdonk,M. L.; Rees, D. C. Group efficiency: a guideline for hits-to-leads chemistry. ChemMedChem 2008, 3, 1179–1180.

(63) Taber, D. F.; Korsmeyer, R.W. Simple stereoselective synthesis of(()-oplopanone. J. Org. Chem. 1978, 43, 4925–4927.

(64) Mutsukado, M.; Tanikawa, K.; Shikada, K.; Sakoda, R. 3(2H)-Pyridazinones and Antiallergic Agents Containing Them.EP193853, 1986.

(65) Dalvit, C.; Pevarello, P.; Tato, M.; Veronesi, M.; Vulpetti, A.;Sundstrom, M. Identification of compounds with binding affinityto proteins via magnetization transfer from bulk water. J Biomol.NMR 2000, 18, 65–68.

(66) Leslie, A. G. W.; Brick, P.; Wonacott, A. MOSFLM. DaresburyLab. Inf. Q. Protein Crystallogr. 2004, 18, 33–39.

(67) Collaborative Computational Project, No. 4. The CCP4 suite:programs for protein crystallography. Acta Crystallogr. 1994,D50, 760-763.

(68) Stebbins, C. E.; Russo, A. A.; Schneider, C.; Rosen, N.; Hartl,F. U.; Pavletich,N. P. Crystal structure of anHsp90-geldanamycincomplex: targeting of a protein chaperone by an antitumor agent.Cell 1997, 89, 239–250.

(69) Obermann, W. M.; Sondermann, H.; Russo, A. A.; Pavletich,N. P.; Hartl, F. U. In vivo function of Hsp90 is dependenton ATP binding and ATP hydrolysis. J. Cell Biol. 1998, 143,901–910.

(70) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Refinement ofmacromolecular structures by the maximum-likelihood method.Acta Crystallogr. D 2004, D53, 240–255.

(71) Roversi, P.; Blanc, E.; Vonrhein, C.; Evans, G.; Bricogne, G.Modelling prior distributions of atoms for macromolecular refine-ment and completion.Acta Crystallogr., Sect. D: Biol. Crystallogr.2000, 56, 1316–1323.

(72) Mooij,W.T.;Hartshorn,M. J.; Tickle, I. J.; Sharff,A. J.; Verdonk,M. L.; Jhoti, H. Automated protein-ligand crystallography forstructure-based drug design. ChemMedChem 2006, 1, 827–838.

(73) Hartshorn, M. J. AstexViewer: a visualisation aid for structure-based drug design. J. Comput.-Aided Mol. Des 2002, 16, 871–881.

(74) Emsley, P.; Cowtan, K. Coot: model-building tools for moleculargraphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60,2126–2132.

(75) Laskowski, R. A. PDBsum: summaries and analyses of PDBstructures. Nucleic Acids Res. 2001, 29, 221–222.

(76) Turnbull, W. B.; Daranas, A. H. On the value of c: can low affinitysystems be studied by isothermal titration calorimetry? J. Am.Chem. Soc. 2003, 125, 14859–14866.

(77) Onuoha, S. C.; Mukund, S. R.; Coulstock, E. T.; Sengerova, B.;Shaw, J.; McLaughlin, S. H.; Jackson, S. E.Mechanistic studies onHsp90 inhibition by ansamycin derivatives. J.Mol. Biol. 2007, 372,287–297.

(78) Sigurskjold, B.W. Exact analysis of competition ligand binding bydisplacement isothermal titration calorimetry. Anal. Biochem.2000, 277, 260–266.

(79) Shao, Y.; Molnar, L. F.; Jung, Y.; Kussmann, J.; Ochsenfeld, C.;Brown, S. T.; Gilbert, A. T.; Slipchenko, L. V.; Levchenko, S. V.;O’Neill,D. P.;DiStasio,R.A., Jr.; Lochan,R.C.;Wang,T.; Beran,G. J.; Besley, N. A.; Herbert, J. M.; Lin, C. Y.; Van Voorhis, T.;Chien, S. H.; Sodt, A.; Steele, R. P.; Rassolov, V. A.;Maslen, P. E.;Korambath, P. P.; Adamson, R. D.; Austin, B.; Baker, J.; Byrd,E. F.; Dachsel, H.; Doerksen, R. J.; Dreuw, A.; Dunietz, B. D.;Dutoi, A. D.; Furlani, T. R.; Gwaltney, S. R.; Heyden, A.; Hirata,S.; Hsu, C. P.; Kedziora, G.; Khalliulin, R. Z.; Klunzinger, P.; Lee,A. M.; Lee, M. S.; Liang, W.; Lotan, I.; Nair, N.; Peters, B.;Proynov, E. I.; Pieniazek, P. A.; Rhee, Y.M.; Ritchie, J.; Rosta, E.;Sherrill, C. D.; Simmonett, A. C.; Subotnik, J. E.; Woodcock,H. L., III; Zhang, W.; Bell, A. T.; Chakraborty, A. K.; Chipman,D. M.; Keil, F. J.; Warshel, A.; Hehre, W. J.; Schaefer, H. F., III;Kong, J.; Krylov, A. I.; Gill, P.M.; Head-Gordon,M.Advances inmethods and algorithms in a modern quantum chemistry programpackage. Phys. Chem. Chem. Phys. 2006, 8, 3172–3191.

(80) Verdonk, M. L.; Chessari, G.; Cole, J. C.; Hartshorn, M. J.;Murray, C.W.; Nissink, J. W.; Taylor, R. D.; Taylor, R.Modelingwater molecules in protein-ligand docking using GOLD. J. Med.Chem. 2005, 48, 6504–6515.

(81) Watson, P.; Verdonk, M. L.; Hartshorn, M. J. A Web-basedplatform for virtual screening. J. Mol. Graph. Modell. 2003, 22,71–82.

(82) Jones, G.; Willett, P.; Glen, R. C. Molecular recognition ofreceptor sites using a genetic algorithm with a description ofdesolvation. J. Mol. Biol. 1995, 245, 43–53.

(83) Eldridge,M.D.;Murray, C.W.; Auton, T. R.; Paolini, G. V.;Mee,R. P. Empirical scoring functions. 1. The development of a fastempirical scoring function to estimate the binding affinity ofligands in receptor complexes. J. Comput.-Aided Mol. Des. 1997,11, 425–445.

(84) Verdonk, M. L.; Cole, J. C.; Hartshorn, M.; Murray, C. W.;Taylor, R. D. Improved protein-ligand docking using GOLD.Proteins 2003, 52, 609–623.

(85) Squires, M. S.; Feltell, R. E.; Wallis, N. G.; Lewis, E. J.; Smith,D. M.; Cross, D. M.; Lyons, J. F.; Thompson, N. T. Biologicalcharacterization of AT7519, a small-molecule inhibitor of cyclin-dependent kinases, in human tumor cell lines. Mol. Cancer Ther.2009, 8, 324–332.

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